Polypeptide porous body and method for producing same

ABSTRACT

A polypeptide porous body of the present invention is a porous body of a polypeptide derived from spider silk proteins. The polypeptide includes a water-insoluble polypeptide. The polypeptide porous body has an apparent density of 0.1 g/cm 3  or less. A method for producing the polypeptide porous body includes: a solution production step in which the polypeptide is dissolved in at least one solvent selected from DMSO, DMF, and these with an inorganic salt, so as to obtain a solution of the polypeptide; a step in which the solution produced in the solution production step is substituted with a water-soluble solvent so as to obtain a polypeptide gel; and a step in which the polypeptide gel is dried. Thereby, the present invention provides a polypeptide porous body having excellent water absorbability and a polypeptide porous body suitable for application to a living body, and a method for producing the same.

TECHNICAL FIELD

The present invention relates to a polypeptide porous body made from apolypeptide derived from spider silk proteins, and a method forproducing the same.

BACKGROUND ART

A polypeptide hydrogel is used as a biomaterial for artificialcartilage, etc. Patent Document 1 proposes a hydrogel, a foam, and thelike obtained by dissolving silk fibroin in a hygroscopic polymer suchas polyethylene glycol. Patent Document 2 discloses a photocrosslinkedgel made from spider silk.

PRIOR ART DOCUMENTS Patent Documents

Patent Document 1: JP 2007-515391 A

Patent Document 2: JP 2008-506409 A

DISCLOSURE OF INVENTION Problem to be Solved by the Invention

However, an urea aqueous solution, which is a conventionally proposedsolvent, does not have a sufficient power to dissolve spider silkproteins, and a guanidine aqueous solution, hexafluoroisopropanol(HFIP), and the like are expensive and difficult to be applied to aliving body if they remain in a product. Further, the waterabsorbability of conventional silk fibroin foams is unknown.

To solve the above conventional problems, the present invention providesa polypeptide porous body having excellent water absorbability and apolypeptide porous body suitable for application to a living body, and amethod for producing the same.

Means for Solving Problem

A polypeptide porous body of the present invention is a porous body of apolypeptide derived from spider silk proteins, wherein the polypeptideincludes a water-insoluble polypeptide, and the polypeptide porous bodyhas an apparent density of 0.1 g/cm³ or less.

A method for producing a polypeptide porous body of the presentinvention includes: a solution production step in which a polypeptidederived from spider silk proteins is dissolved in at least onedissolving solvent selected from the group consisting of: (A) dimethylsulfoxide; (B) dimethyl sulfoxide with an inorganic salt; and (C) N,N-dimethylformamide with an inorganic salt, so as to obtain a solutionof the polypeptide; a step in which the solution of the polypeptideproduced in the solution production step is substituted with awater-soluble solvent so as to obtain a polypeptide gel; and a step inwhich the polypeptide gel is dried. Incidentally, “a polypeptide isdissolved in a dissolving solvent” used herein includes both of a statein which a polypeptide is completely dissolved in a dissolving solventand a state in which polypeptide microparticles are dispersed in adissolving solvent and thus are substantially dissolved in thedissolving solvent. Hereinafter, the expression shall have the samemeaning as described above.

Effect of the Invention

In the present invention, by using a specific solvent in the productionof the polypeptide solution, substituting the solution with awater-soluble solvent to obtain a polypeptide gel, and drying theobtained gel, a polypeptide porous body can be produced in which theamount of the remaining solvent is little or the amount of the remainingsolution is sufficiently low, whereby a polypeptide porous body suitablefor application to a living body can be provided. Further, since theapparent density of the polypeptide porous body according to the presentinvention is sufficiently low, a void volume inside the porous body islarge. Thus, the polypeptide porous body exhibits high waterabsorbability. The porous body is also called as a xerogel, which has amesh structure having voids in a solid phase skeleton. Additionally, thesolvents used in the present invention are those that have been used inthe production of acrylic fibers and polyimid, and they are low cost.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an optical photograph of a polypeptide porous body in oneexample of the present invention.

FIG. 2 is a photograph of the polypeptide porous body in the example ofthe present invention taken by a scanning electron microscope (SEM) at70× magnification.

FIG. 3 is a photograph of the same at 200× magnification.

FIG. 4 is a photograph of the same at 1,000× magnification.

DESCRIPTION OF THE INVENTION

A polypeptide derived from spider silk proteins is used as the proteinof the present invention. The polypeptide derived from spider silkproteins is not limited particularly as long as it is derived from orsimilar to natural type spider silk proteins. Examples of thepolypeptide derived from spider silk proteins include variants, analogs,derivatives, and the like of natural type spider silk proteins. In termsof excellent tenacity, the recombinant spider silk protein preferably isa recombinant spider silk protein derived from major dragline silkproteins produced in major ampullate glands of spiders. Examples of themajor dragline silk proteins include major ampullate spidroins MaSp1 andMaSp2 derived from Nephila clavipes, and ADF3 and ADF4 derived fromAraneus diadematus, etc.

The recombinant spider silk protein may be a recombinant spider silkprotein derived from minor dragline silk produced in minor ampullateglands of spiders. Examples of the minor dragline silk proteins includeminor ampullate spidroins MiSp1 and MiSp2 derived from Nephila clavipes.

Other than these, the recombinant spider silk protein may be arecombinant spider silk protein derived from flagelliform silk proteinsproduced in flagelliform glands of spiders. Examples of the flagelliformsilk proteins include flagelliform silk proteins derived from Nephilaclavipes, etc.

Examples of the polypeptide derived from major dragline silk proteinsinclude a polypeptide containing two or more units of an amino acidsequence represented by the formula 1: REP1−REP2 (1), preferably apolypeptide containing four or more units thereof, and more preferably apolypeptide containing six or more units thereof. In the polypeptidederived from major dragline silk proteins, units of the amino acidsequence represented by the formula (1): REP1−REP2 (1) may be the sameor different from each other. In the formula (1), the REP1 representspolyalanine. In the REP1, the number of alanine residues arranged insuccession is preferably 2 or more, more preferably 3 or more, furtherpreferably 4 or more, and particularly preferably 5 or more. Further, inthe REP1, the number of alanine residues arranged in succession ispreferably 20 or less, more preferably 16 or less, further preferably 14or less, and particularly preferably 12 or less. In the formula (1), theREP2 is an amino acid sequence composed of 10 to 200 amino acidresidues. The total number of glycine, serine, glutamine, proline, andalanine residues contained in the amino acid sequence is 40% or more,preferably 50% or more, and more preferably 60% or more with respect tothe total number of amino acid residues contained therein.

In the major dragline silk, the REP1 corresponds to a crystal region ina fiber where a crystal β sheet is formed, and the REP2 corresponds toan amorphous region in a fiber where most of the parts lack regularstructures and that has more flexibility. Further, the [REP1−REP2]corresponds to a repetitious region (repetitive sequence) composed ofthe crystal region and the amorphous region, which is a characteristicsequence of dragline silk proteins.

An example of the polypeptide containing two or more units of the aminoacid sequence represented by the formula 1: REP1−REP2 (1) is arecombinant spider silk protein derived from ADF3 having an amino acidsequence represented by any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,and SEQ ID NO: 4. The amino acid sequence represented by SEQ ID NO: 1 isan amino acid sequence from the 1st residue to the 631st residue of anamino acid sequence that is obtained by adding an amino acid sequence(SEQ ID NO: 5) composed of a start codon, an His 10-tag and an HRV3CProtease (Human rhinovirus 3C Protease) recognition site to theN-terminal of a partial amino acid sequence of ADF3 obtained from theNCBI database (NCBI Genebank Accession No.: AAC47010, GI: 1263287). Theamino acid sequence represented by SEQ ID NO: 2 is an amino acidsequence obtained by the following mutation: in an amino acid sequenceof ADF3 (NCBI Genebank Accession No.: AAC47010, GI: 1263287) to theN-terminal of which has been added the amino acid sequence (SEQ ID NO:5) composed of a start codon, an His 10-tag and an HRV3C Protease (Humanrhinovirus 3C Protease) recognition site, 1st to 13th repetitive regionsare about doubled and the translation ends at the 1154th amino acidresidue. The amino acid sequence represented by SEQ ID NO: 3 is an aminoacid sequence obtained by adding the amino acid sequence (SEQ ID NO: 5)composed of a start codon, an His 10-tag and an HRV3C Protease (Humanrhinovirus 3C Protease) recognition site, to the N-terminal of a partialamino acid sequence of ADF3 (NCBI Genebank Accession No.: AAC47010, GI:1263287) obtained from the NCBI database. The amino acid sequencerepresented by SEQ ID NO: 4 is an amino acid sequence obtained asfollows: in an amino acid sequence of ADF3 (NCBI Genebank Accession No.:AAC47010, GI: 1263287) to the N-terminal of which has been added theamino acid sequence (SEQ ID NO: 5) composed of a start codon, an His10-tag and an HRV3C Protease (Human rhinovirus 3C Protease) recognitionsite, 1st to 13th repetitive regions are about doubled. Further, thepolypeptide containing two or more units of the amino acid sequencerepresented by the formula 1: REP1−REP2 (1) may be a polypeptide that iscomposed of an amino acid sequence represented by any of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 in which one or a pluralityof amino acids have been substituted, deleted, inserted and/or added andthat has repetitious regions composed of crystal regions and amorphousregions.

In the present invention, “one or a plurality of” refers to 1 to 40, 1to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, or 1 or a few, forexample. Further, in the present invention, “one or a few” refers to 1to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

An example of the recombinant spider silk protein derived from minordragline silk proteins is a polypeptide containing an amino acidsequence represented by the formula 2: REP3 (2). In the formula 2, theREP 3 indicates an amino acid sequence composed of(Gly-Gly-Z)m(Gly-Ala)l(A)r, where Z indicates any one of amino acids, inparticular, it preferably is an amino acid selected from the groupconsisting of Ala, Tyr and Gln. Further, preferably, m is 1 to 4, 1 is 0to 4, and r is 1 to 6.

Among spider silks, the minor dragline silk is wound spirally from thecenter of a spider net, and used as a reinforcement of the net and ayarn to wrap a captured prey. The minor dragline silk is inferior to themajor dragline silk in tensile strength, but is known to have highstretchability. The reason for this is considered to be as follows: inthe minor dragline silk, since many crystal regions are formed ofregions where glycine and alanine are arranged alternately insuccession, hydrogen bonds in the crystal regions weaken easily ascompared with the major dragline silk whose crystal regions are formedonly of alanine.

Examples of the recombinant spider silk protein derived fromflagelliform silk proteins include a polypeptide containing an aminoacid sequence represented by the formula 3: REP4 (3). In the formula 3,the REP 4 indicates an amino acid sequence composed of(Gly-Pro-Gly-Gly-X)n, where X indicates any one of amino acids, inparticular, it preferably is an amino acid selected from the groupconsisting of Ala, Ser, Tyr and Val. Further, n indicates a number of 4or larger, preferably 10 or larger, and more preferably 20 or larger.

Among spider silks, the flagelliform silk does not have crystal regionsbut has repetitious regions composed of amorphous regions, which is amajor characteristic of the flagelliform silk. It is considered thatsince the major dragline silk and the like have repetitious regionscomposed of crystal regions and amorphous regions, they have both ofhigh strength and stretchability. Meanwhile, regarding the flagelliformsilk, the strength is inferior to that of the major dragline silk butthe stretchability is high. The reason for this is considered to be thatthe flagelliform silk is composed mostly of amorphous regions.

The polypeptide can be produced using a host that has been transformedby an expression vector containing a gene encoding a polypeptide. Amethod for producing a gene is not limited particularly, and it may beproduced by amplifying a gene encoding a natural type spider silkprotein from a cell containing the desired gene of spider by apolymerase chain reaction (PCR), etc., and cloning it, or may besynthesized chemically. A method for chemically synthesizing a gene alsois not limited particularly, and it can be synthesized as follows, forexample: based on information of amino acid sequences of natural typespider silk proteins obtained from the NCBI web database,oligonucleotides that have been synthesized automatically with AKTAoligopilot plus 10/100 (GE Healthcare Japan Corporation) are linked byPCR, etc. At this time, in order to facilitate purification andobservation of protein, a gene may be synthesized that encodes a proteincomposed of an amino acid sequence in which an amino acid sequencecomposed of a start codon and an His 10-tag has been added to theN-terminal of the amino acid sequence. Examples of the expression vectorinclude a plasmid, a phage, a virus, and the like that can expressprotein based on a DNA sequence. The plasmid-type expression vector isnot limited particularly as long as it allows a target gene to beexpressed in a host cell and it can amplify itself. For example, in thecase of using Escherichia coli Rosetta (DE3) as a host, a pET22b(+)plasmid vector, a pCold plasmid vector, and the like can be used. Amongthese, in terms of productivity of protein, the use of the pET22b(+)plasmid vector is preferred. Examples of the host include animal cells,plant cells, microbes, etc.

Further, as the polypeptide, a polypeptide that can form a gel uponcontact with a water-soluble solvent is used. An example of thepolypeptide that can form a gel is a polypeptide that aggregates easily(e.g., a polypeptide having a relatively large molecular weight) whenthe solution produced in the solution production step is substitutedwith a water-soluble solvent. Incidentally, even if a polypeptide doesnot have such an easy aggregation property, for example, by increasingthe concentration of the polypeptide in the solution produced in thesolution production step, the polypeptide aggregates when the solutionis substituted with a water-soluble solvent. Thus, a polypeptide gel isformed. The polypeptide also may aggregate by lowering the temperaturefrom a high-temperature state to a low-temperature state. In otherwords, a polypeptide porous body is produced using a polypeptide thataggregates and forms a gel when the solution produced in the solutionproduction step is substituted with a water-soluble solvent. Thepolypeptide porous body includes such a polypeptide.

The polypeptide porous body has continuous pores. It is believed thatthe continuous pores are formed by through holes of water desorbed inthe vacuum freeze-drying step. As a result, a xerogel is formed, whichhas a mesh structure having voids in a solid phase skeleton. Such apolypeptide porous body has an apparent density of 0.1 g/cm³ or less,preferably in a range from 0.01 to 0.08 g/cm³. When the apparent densityis less than 0.01 g/cm³, the void volume will be excessive because theapparent density is too small, which may decrease the strength of thepolypeptide porous body.

The maximum water absorption ratio of the porous body is preferably twotimes or more, and more preferably three times or more, in terms ofweight. The upper limit thereof is preferably 20 times or less, and morepreferably 18 times or less. The maximum water absorption ratioindicates a point at which the porous body absorbs water to the maximumand does not leak water by inclination.

The polypeptide porous body of the present invention is produced by: asolution production step in which a polypeptide derived from spider silkproteins is dissolved in at least one dissolving solvent selected fromthe group consisting of (A) dimethyl sulfoxide; (B) dimethyl sulfoxidewith an inorganic salt; and (C) N, N-dimethylformamide with an inorganicsalt, so as to obtain a solution of the polypeptide; a step in which thesolution of the polypeptide produced in the solution production step issubstituted with a water-soluble solvent so as to obtain a polypeptidegel; and a step in which the polypeptide gel is dried. The polypeptideporous body can be formed into a predetermined shape by performing amolding step in which the solution is poured into a mold between thesolution production step and the solvent substitution step using awater-soluble solvent, or by cutting the gel after the solventsubstitution step using a water-soluble solvent. Further, at least oneselected from the group consisting of dimethyl sulfoxide and N,N-dimethylformamide may be present inside the obtained polypeptideporous body. The amount of the dissolving solvent present therein is notlimited particularly, and it is an amount that remains thereinunintentionally after the solution produced in the solution productionstep is substituted with the water-soluble solvent.

In addition to the substances indicated in (A)-(C) above, the dissolvingsolvent may contain alcohol and/or water. The dissolving solvent is apolar solvent, and tends to absorb moisture in air. Therefore, in somecases, commercial solvents contain several % of water. The dissolvingsolvent may contain this amount of water and/or alcohol. Incidentally,the substances functioning as the dissolving solvent are those indicatedin (A)-(C) above.

The water-soluble solvent refers to a solvent containing water. Examplesof the water-soluble solvent include water, a water-soluble buffersolution, and saline.

In terms of high compatibility with the human body, preferably, thewater-soluble solvent is water. Although the water is not limitedparticularly, it may be pure water, distilled water, ultrapure water,etc.

The viscosity of the solution after the solution production step ispreferably 5 to 80 cP (centipoises), and more preferably 10 to 50 cP.Within the above range, favorable handleability and convenience will beobtained.

In the present invention, a solvent containing DMSO and/or DMF (polarsolvent) is used as the solvent. DMSO has a melting point of 18.4° C.and a boiling point of 189° C. DMF has a melting point of −61° C. and aboiling point of 153° C. DMSO and DMF have much higher boiling pointsthan hexafluoroisopropanol (HFIP) and hexafluoroacetone (HFAc) havingboiling points of 59° C. and −26.5° C., respectively, which have beenused in conventional methods, and hence DMSO and DMF have favorabledissolubility. Further, in view of the fact that DMSO and DMF have beenused also in general industrial fields for acrylic fiber polymerization,acrylic fiber spinning solutions, and solvents for polyimidepolymerization, they are low-cost substances with proven safety.

Addition of an inorganic salt to DMSO or DMF further increases thesolubility of a solute. The inorganic salt is at least one selected fromalkali metal halides (e.g., LiCl, LiBr, etc), alkaline-earth metalhalides (e.g., CaCl₂), alkaline-earth metal nitrate (e.g., Ca(NO₃)₂,etc.), and sodium thiocyanate (e.g., NaSCN, etc.). When the dissolvedcomponents are assumed to be 100 mass %, the percentage of the inorganicsalt preferably ranges from 0.1 to 20 mass %.

The polypeptide gel is produced by substituting the solvent with thewater-soluble solvent. At this time, the polypeptide gel is notdissolved in water. Preferably, the step of substituting the solventwith the water-soluble solvent is performed in the following manner: thesolution of the polypeptide obtained by dissolving the polypeptide inthe solvent is placed in a dialysis membrane, the dialysis membrane isimmersed in a water-soluble solvent, and the water-soluble solvent isrenewed at least one time. Specifically, preferably, the step ofsubstituting the solvent with the water-soluble solvent is performed byplacing the solution after the solution production step in a dialysismembrane, leaving it to stand for 3 hours in a water-soluble solvent inan amount 100 times or more the amount of the solution (one batch), andrenewing the water-soluble solvent three or more times in total. Anydialysis membrane that does not allow the polypeptide in the solution topass therethrough can be used. An example of the dialysis membrane is acellulose dialysis membrane. By repeating the substitution using thewater-soluble solvent, the amount of the dissolving solvent can bereduced close to zero. In the latter half of the desolvation step, it isunnecessary to use a dialysis membrane.

The amount of the solvent, i.e., dimethyl sulfoxide (DMSO) or N,N-dimethylformamide (DMF), remaining in the polypeptide after thesubstitution step using the water-soluble solvent can be measured by anuclear magnetic resonance spectrometer (NMR). A1,2-dichloroethane-formic acid solution can be used as an internalstandard.

In the drying step, preferably, vacuum freeze-drying is adopted. Thedegree of vacuum at vacuum freeze-drying is preferably 200 Pa or less,more preferably 150 Pa or less, and further preferably 100 Pa or less.By vacuum drying, water evaporates from the polypeptide gel, and thetemperature declines by the evaporation latent heat, whereby it isbrought into a frozen state. The temperature of the polypeptide atvacuum freeze-drying is preferably 70° C. or less, more preferably 60°C. or less, and further preferably 50° C. or less. Incidentally, priorto vacuum freeze-drying, the polypeptide gel may be pre-frozen at atemperature of −10° C. to −45° C. for about 10 to 36 hours. The moisturecontent after freeze-drying is preferably 5.0% or less, and morepreferably 3.0% or less.

EXAMPLES

Hereinafter, the present invention will be described in further detailby way of examples. Note that the present invention is not limited tothe following examples. Water is used as the water-soluble solvent inthe examples.

<Methods of Various Measurements>

(1) Measurement of Remaining Amount of Solvent

As an internal standard, a 1,2-dichloroethane-formic acid solution at aconcentration of 3,100 ppm (0.00310 mg/ml) was prepared. 500 μl of aprotein solution (obtained by dissolving 0.1 g of a polypeptide porousbody in 10 ml of formic acid) and 500 μl of an internal standardsolution were mixed. For H-NMR measurement, an acetonitrile deuteratedsolvent was added to the mixed solution in an amount approximatelyequivalent to that of the mixture solution so as to dilute the solutionto about two times. Then, H-NMR measurement was performed (NMR model:JNM-ECX 100 manufactured by JOEL Ltd.). The H-NMR integrated intensityof 1,2-dichloroethane (internal standard sample) was compared with theH-NMR integrated intensity of DMSO. A calibration curve was formed bypreparing a DMSO-formic acid solution at 3 ppm to 3000 ppm and followingthe above-mentioned protocol. By comparison with the calibration curve,the concentration of DMSO in the protein solution was calculated. Anuclear magnetic resonator (NMR) manufactured by JOEL Ltd. was used forthe measurement of the concentration of DMSO.

(2) Viscosity

An EMS machine manufactured by Kyoto Electronics Manufacturing Co., Ltd.was used.

Example 1 1. Preparation of Polypeptide

<Gene Synthesis of ADF3Kai-A>

A partial amino acid sequence of ADF3, which is one of two principaldragline silk proteins of Araneus diadematus, was obtained from the NCBIweb database (NCBI Accession No.: AAC47010, GI: 1263287), and an aminoacid sequence (SEQ ID NO: 5) composed of a start codon, an His 10-tagand an HRV3C Protease (Human rhinovirus 3C Protease) recognition sitewas added to the N-terminal of the partial amino acid sequence of ADF3,so as to synthesize a gene encoding a polypeptide (ADF3Kai-A) composedof an amino acid sequence (SEQ ID NO: 1), i.e., the 1st residue to the631st residue from the N-terminal of the resultant sequence.Consequently, a pUC57 vector to which a gene of ADF3Kai-A composed of abase sequence represented by SEQ ID NO: 6 had been introduced wasobtained (having an Nde I site immediately upstream of the 5′ terminalof the gene and an Xba I site immediately downstream of the 5′ terminalthereof). Thereafter, the gene was subjected to a restriction enzymetreatment with Nde I and EcoR I, and recombined into a pET22b(+)expression vector. Thus, a pET22b(+) vector to which the gene ofADF3Kai-A had been introduced was obtained.

<Expression of Protein>

The obtained pET22b(+) expression vector containing the gene sequence ofADF3Kai-A was transformed into Escherichia coli Rosetta (DE3). Theobtained single colony was incubated for 15 hours in 2 mL of an LBculture medium containing ampicillin. Thereafter, 1.4 ml of said culturesolution was added to 140 mL of an LB culture medium containingampicillin, and incubated to an OD₆₀₀ of 3.5 under the conditions of 37°C. and 200 rpm. Next, the culture solution with the OD₆₀₀ of 3.5 wasadded to 7 L of a 2×YT culture medium containing ampicillin, togetherwith 140 mL of 50% glucose, and incubated further to the OD₆₀₀ of 4.0.Thereafter, isopropyl-β-thiogalactopyranoside (IPTG) was added to theobtained culture solution with the OD₆₀₀ of 4.0 so that the fmalconcentration would be 0.5 mM, thereby inducing the expression ofprotein. After a lapse of two hours from the addition of IPTG, theculture solution was centrifuged and bacterial cells were collected.Protein solutions prepared from the culture solution before the additionof IPTG and the culture solution after the addition of IPTG were eachelectrophoresed in a polyacrylamide gel. Consequently, a target bandsize (about 56.1 kDa) was observed with the addition of IPTG, and theexpression of the target protein (ADF3Kai-A) was confirmed.

Purification

(1) About 50 g of bacteria cells of the Escherichia coli expressing theADF3Kai-A protein and 300 ml of a buffer solution M (20 mM Tris-HCl, pH7.4) were placed in a centrifuge tube (1000 ml). After dispersing thebacteria cells with a mixer (“T18 basic ULTRA TURRAX” manufactured byIKA, level 2), the dispersion was centrifuged (11,000 g, 10 minutes,room temperature) with a centrifuge (“Model 7000” manufactured by KubotaCorporation), and a supernatant was discarded.

(2) To a precipitate (bacteria cells) obtained by the centrifugation,300 ml of the buffer solution M and 3 ml of 0.1 M PMSF (dissolved byisopropanol) were added. After dispersing the precipitate for 3 minuteswith the above mixer (level 2) manufactured by IKA, the bacteria cellswere disrupted repeatedly for three times using a high-pressurehomogenizer (“Panda Plus 2000” manufactured by GEA Niro Soavi).

(3) To the disrupted bacterial cells, 300 mL of a buffer solution B (50mM Tris-HCL, 100 mM NaCl, pH 7.0) containing 3 w/v % of SDS was added.After dispersing well the bacterial cells with the above mixer (level 2)manufactured by IKA, the dispersion was stirred for 60 minutes with ashaker (manufactured by TAITEC CORPORATION, 200 rpm, 37° C.).Thereafter, the stirred dispersion was centrifuged (11,000 g, 30minutes, room temperature) with the above centrifuge manufactured byKubota Corporation, and a supernatant was discarded, whereby SDS washinggranules (precipitate) were obtained.

(4) The SDS washing granules were suspended in a DMSO solutioncontaining 1M lithium chloride so that the concentration would be 100mg/mL, and heat-treated for 1 hour at 80° C. Thereafter, the heatedsuspension was centrifuged (11,000 g, 30 minutes, room temperature) withthe above centrifuge manufactured by Kubota Corporation, and asupernatant was collected.

(5) Ethanol in an amount three times greater than that of the collectedsupernatant was prepared. The collected supernatant was added to theethanol, and left to stand for 1 hour at room temperature. Thereafter,the resultant was centrifuged (11,000 g, 30 minutes, room temperature)with the above centrifuge manufactured by Kubota Corporation to collectaggregated protein. Next, a process of washing aggregated protein usingpure water and collecting aggregated protein by centrifugation wasrepeated three times, and thereafter water was removed by a freeze dryerto collect freeze-dried powder. The purification degree of the targetprotein ADF3Kai-A (about 56.1 kDa) in the obtained freeze-dried powderwas checked by analyzing images of the results of polyacrylamide gelelectrophoresis (CBB staining) of said protein powder using Totallab(Nonlinear Dynamics). As a result, the purification degree of ADF3Kai-Awas about 85%.

2. Adjustment of Solution

0.8 g of spider silk protein powder (ADF3Kai-A) was added to 20 ml ofDMSO (containing 1M LiCl), followed by dissolution at 80° C. for 30minutes. Then, dusts and bubbles were removed from the solution. Theviscosity of the solution was 30.8 cP (centipoises). The solution wasplaced a dialysis tube (Cellulose Tube 36/32 manufactured by SankoJunyaku Co., Ltd. (presently EIDIA Co., Ltd)).

3. Substitution with Water

The dialysis tube was placed in a beaker filled with 3 L of pure water,left to stand for 3 hours, and water was renewed. This operation wasrepeated six times in total. Thus, the spider silk protein in thesolution aggregated, and a hydrogel in which almost all of DMSO wassubstituted with water was produced. The moisture content of theobtained gel was 95.3 mass %.

4. Vacuum Freeze-Drying

The hydrogel was freeze-dried by a freeze dryer (“FDU-1200” manufacturedby Tokyo Rikakiki Co., Ltd.) under conditions of 14 Pa and −45° C. for15 hours.

5. Results

(1) The amount of the solvent remaining in the obtained porous body was2.63 g based on 100 g of the porous body.

(2) The apparent density of the obtained porous body was 0.077 g/cm³.Incidentally, the apparent density was measured in accordance with aknown technique. The densities (apparent densities) of Examples 2-4described below also were measured in the same manner as in Example 1.

(3) The maximum water absorption ratio of the obtained porous body was15.4 times. This water absorption ratio indicates a point at which theporous body absorbs water to the maximum and does not leak water byinclination. The same applies to the following examples.

(4) FIGS. 1-4 show the obtained porous body.

Example 2

A gel of Example 2 was produced in the same manner as in Example 1 usingthe same polypeptide as that of Example 1. A 20 mg/ml dope (DMSO+1MLiCl) was prepared using 0.4 g of the polypeptide powder so as toproduce a gel with a low concentration. The viscosity of the dope was13.9 cP. The moisture content of the obtained gel was 98.8 mass %. Thegel was subjected to vacuum freeze-drying under the same condition as inExample 1. The density of the obtained porous body was 0.020 g/cm³. Themaximum water absorption ratio of the obtained porous body was 7.2times.

Example 3

A gel of Example 3 was produced in the same manner as in Example 1 usingthe same polypeptide as that of Example 1. A 20 mg/ml dope (DMSO withoutsalt) was prepared using 0.4 g of the polypeptide powder so as toproduce a gel. The moisture content of the obtained gel was 97.4 mass %.The gel was subjected to vacuum freeze-drying under the same conditionas in Example 1. The density of the obtained porous body was 0.036g/cm³. The maximum water absorption ratio of the obtained porous bodywas 6.1 times.

Example 4

A gel of Example 4 was produced in the same manner as in Example 1 usingthe same polypeptide as that of Example 1. A 20 mg/ml dope (DMF+1M LiCl)was prepared using 0.4 g of the polypeptide powder so as to produce agel. The moisture content of the obtained gel was 98.2 mass %. The gelwas subjected to vacuum freeze-drying under the same condition as inExample 1. The density of the obtained porous body was 0.039 g/cm³. Themaximum water absorption ratio of the obtained porous body was 3.8times.

Although water was used in the substitution step in the above Examples1-4, the same effect can be obtained using other water-soluble solvents.

INDUSTRIAL APPLICABILITY

The polypeptide porous body of the present invention is useful as aslow-release substrate such as a medicament and perfume, an absorber,etc.

SEQUENCE LISTING FREE TEXT

SEQ ID NOS: 1-5 amino acid sequences

SEQ ID NO: 6 base sequence

1. A polypeptide porous body that is a porous body of a polypeptidederived from spider silk proteins, wherein the polypeptide includes awater-insoluble polypeptide, and the polypeptide porous body has anapparent density of 0.1 g/cm³ or less.
 2. The polypeptide porous bodyaccording to claim 1, wherein at least one selected from the groupconsisting of dimethyl sulfoxide and N,N-dimethylformamide is presentinside the polypeptide porous body.
 3. The polypeptide porous bodyaccording to claim 1, wherein the polypeptide porous body has anapparent density of 0.01 to 0.08 g/cm³.
 4. The polypeptide porous bodyaccording to claim 1, wherein the polypeptide porous body has a waterabsorption ratio of 2 to 20 times.
 5. A method for producing apolypeptide porous body, comprising: a solution production step in whicha polypeptide derived from spider silk proteins is dissolved in at leastone dissolving solvent selected from the group consisting of: (A)dimethyl sulfoxide; (B) dimethyl sulfoxide with an inorganic salt; and(C) N, N-dimethylformamide with an inorganic salt, so as to obtain asolution of the polypeptide; a step in which the solution of thepolypeptide produced in the solution production step is substituted witha water-soluble solvent so as to obtain a polypeptide gel; and a step inwhich the polypeptide gel is dried.
 6. The method for producing apolypeptide porous body according to claim 5, wherein the polypeptideporous body is produced using an aqueous solution that is obtained bysubstituting the solution of the polypeptide with water.
 7. The methodfor producing a polypeptide porous body according to claim 5, whereinthe substitution step using the water-soluble solvent is a step in whichthe solution of the polypeptide obtained by dissolving the polypeptidein the dissolving solvent is placed in a dialysis membrane, the dialysismembrane is immersed in a water-soluble solvent, and the water-solublesolvent is renewed at least one time.
 8. The method for producing apolypeptide porous body according to claim 5, wherein the drying isvacuum freeze-drying